Adult skeletal muscle exhibits a remarkable ability to repair and regenerate after trauma or injury. The regenerative capacity of skeletal muscle is due to a reservoir of satellite cells located under the basal lamina and in close proximity to the myofiber sarcolemma. These cells remain quiescent in healthy uninjured muscle, but are rapidly activated in response to muscle damage, exercise, or disease.
Upon activation, satellite cells proliferate and differentiate down the myogenic pathway and are able to repair damaged muscle. Models suggest a subpopulation of satellite cells remain as stem cells to replace activated cells that have progressed down the myogenic lineage pathway. During the activation period, satellite cells express the transcription factors Pax3, Pax7, MyoD, myogenin, and MRF4 as they progress through a developmental program towards muscle repair.
Muscular dystrophy is a term used to refer to a group of genetic disorders that lead to progressive muscle weakness. Muscular dystrophy can result in skeletal muscle weakness and defects in skeletal muscle proteins, leading to a variety of impaired physiological functions. No satisfactory treatment of muscular dystrophy exists. Existing treatments typically focus on ameliorating the effects of the disease and improving the patient's quality of life, such as through physical therapy or through the provision of orthopedic devices.
Mutated genes associated with muscular dystrophy are responsible for encoding a number of proteins associated with the costameric protein network. Such proteins include laminin-2, collagen, dystroglycan, integrins, caveolin-3, ankyrin, dystrophin, α-dystrobrevin, vinculin, plectin, BPAG1b, muscle LIM protein, desmin, actinin-associated LIM protein, α-actin, titin, telethonin, cypher, myotilin, and the sarcoglycan/sarcospan complex.
The most common form of muscular dystrophy, Duchenne muscular dystrophy, is caused by a mutation in the gene responsible for production of dystrophin. Dystrophin is a protein involved in binding cells to the extracellular matrix, including the basement membrane. Congenital muscular dystrophies are caused by gene mutations affecting the production of other costameric proteins. For example, in populations of European descent, the most prevalent congenital muscular dystrophy is caused by a mutation resulting in a lack of α7β1 integrin expression. Like dystrophin, α7β1 integrin is involved in binding cells to the extracellular matrix.
To some extent, a defect in the gene encoding for one of dystrophin or α7β1 integrin is often compensated for by enhanced expression of the other, or another costameric protein, such as utrophin (an analog of dystrophin). Dystrophin, α7β1 integrin, and utrophin all serve as receptors for laminin, which serves as the link to the extracellular matrix. Defective production of laminin-2 itself gives rise to merosin-deficient congenital muscular dystrophy (MCMD) or congenital muscular dystrophy type 1A (MDC1A).
Laminin is a major component of the basement membrane. At least fifteen laminin protein trimers have been identified, each a heterotrimer including an α, β, and γ chain. Laminin is associated with a number of physiological functions, including cell attachment, gene expression, tyrosine phosphorylation of proteins, cell differentiation, as well as cell shape and movement. Laminin is known to bind to cell membranes through integrin receptors. In addition, laminin-2 binds to α-dystroglycan as part of the dystrophin-glycoprotein complex.
The α7β1 integrin is a major laminin receptor expressed in skeletal muscle. The α7β1 integrin plays an important role in the development of neuromuscular and myotendinous junctions. In the adult, the α7β1 integrin is concentrated at junctional sites and found in extrajunctional regions where it mediates the adhesion of the muscle fibers to the extracellular matrix. Mice that lack the α7 chain develop muscular dystrophy that affects the myotendinous junctions. The absence of α7 integrin results in defective matrix deposition at the myotendinous junction. Loss of the α7 integrin in γ-sarcoglycan mice results in severe muscle pathology. Absence of the α7 integrin in mdx mice also results in severe muscular dystrophy, confirming that the α7β1 integrin serves as a major genetic modifier for Duchenne and other muscular dystrophies.
Mutations in the α7 gene are responsible for muscular dystrophy in humans. A screen of 117 muscle biopsies from patients with undefined muscle disease revealed 3 which lacked the α7 integrin chain and had reduced levels of β1D integrin chain. These patients exhibit delayed developmental milestones and impaired mobility consistent with the role for the α7β1 integrin in neuromuscular and myotendinous junction development and function.
Several lines of evidence suggest the α7 integrin may be important for muscle regeneration. For example, during embryonic development, the α7β1 integrin regulates myoblast migration to regions of myofiber formation. It has been found that MyoD (myogenic determination protein) transactivates α7 integrin gene expression in vitro, which would increase α7 integrin levels in activated satellite cells. Human, mouse and rat myoblast cell lines derived from satellite cells express high levels of α7 integrin. Elevated α7 integrin mRNA and protein are detected in the skeletal muscle of 5 week old mdx mice, which correlates with the period of maximum muscle degeneration and regeneration. In addition, the α7β1 integrin associates with muscle specific α1-integrin binding protein (MIBP), which regulates laminin deposition in C2C12 myoblasts. Laminin provides an environment that supports myoblast migration and proliferation. Finally, enhanced expression of the α7 integrin in dystrophic skeletal muscle results in increased numbers of satellite cells.
To date, many efforts to cure or ameliorate muscular dystrophy involve enhancing expression of various components of the costameric network. However, these approaches, while showing some promise in vitro or in transgenic animals, typically do not demonstrate effective results in humans nor provide methods through which therapy could be accomplished in humans. Such routes of therapy are notoriously difficult to implement.
However, it is also well known that direct administration of proteins, particularly large proteins, is very difficult. For example, large size, high charge, short half life, poor stability, high immunogenicity, and poor membrane permeability can limit the bioavailability of administered proteins. In addition, depending on the route of administration, a subject's natural physiological processes can attack and degrade administered proteins. For example, although laminin is known to play a role in the extracellular matrix, it is a particularly large (typically >600 kD), highly charged molecule and consequently difficulties in its administration to patients would likely have been anticipated. Accordingly, efforts to date have focused on more sophisticated treatments, rather than direct administration of therapeutic substances.